The proposed research focuses on the regulation of cellular thermogenesis in brown adipocytes. Information obtained during the preceding grant period identified an alpha adrenergic component to catecholamine stimulation of respiration in hamster brown fat cells. We now wish to probe the cellular-biochemical mechanisms underlying this alpha adrenergic regulation of respiration. Three types of experiments are proposed. First, experiments are proposed to determine if alpha adrenergic stimulation of brown fat cells is accompanied by uncoupling of mitochondria. Second, experiments are proposed to characterize alpha adrenergic regulation of inositol phospholipid metabolism in brown fat cells. These studies are designed to determine if breakdown of PtdIns (4,5)P to inositol triphosphate and diacyloglycerol is accelerated by alpha adrenergic ligands, is calcium dependent and is antagonized by adenosine or by insulin. Other experiments are designed to determine if diacylglycerol formed from breakdown of PtdIns(4,5)P is converted to phosphatidic acid and if PtdIns(4,5)P is reformed by sequential phosphorylations of PtdIns and PtdIns(4,5)P. Thirdly, experiments have been described to determine if alpha adrenergic stimulation of brown fat cells is accompanied by increases in cytosolic calcium measured from fluorescence of Quin-2 and if such increases in cytosolic calcium are a necessary component of the cells physiological response. Given that the alpha adrenergic component of catecholamine contributes approximately 25-30 of the stimulation of thermogenesis, the proposed studies will provide insight into physiological regulation of this process. This project will also contribute information on hormonal regulation of inositol phospholipids and calcium in a unique cell type not previously investigated.